Antibodies and Fc-fusion biologics have been used as therapeutic molecules for the treatment of various diseases in the past decade. Most antibodies on the market are full length antibodies (e.g., IgGs) because their long half-lives allow for less frequent dosing in patients. See, e.g., Lobo et al., J. Pharm. Sci. 93, 2645-2668 (2004). A full-length IgG is composed of two identical Fab fragments which are connected by the dimeric form of Fc fragments through two identical hinge regions. While the Fab region is responsible for targeting the antigen, the Fc region of IgG has been implicated in the antibody's prolonged survival time in serum via the neonatal Fc receptor (FcRn) recycling pathway. See, e.g., Brambell et al., Nature 203, 1352-1354 (1964) and Raghavan et al., Biochemistry 34, 14649-14657 (1995). The intrinsic association constant for monovalent binding by each Fab is usually referred to as the affinity of the antibody, while the bivalent binding ability of two Fabs in an intact IgG antibody is referred to as the avidity of the antibody. In some cases, the apparent equilibrium binding due to the avidity of IgG can be increased up to 100-fold compared with the affinity of the Fab. See, e.g., Ways et al., Biochem J 216, 423-432 (1983). For therapeutic purposes, however, the bivalency of IgG might not always be necessary or desired. For example, a therapeutic IgG would not take advantage of avidity if the targets are monomeric soluble molecules. Additionally, if the targets are multimeric soluble molecules, the dimeric nature of IgG can result in formation of a cross-linked network in plasma leading to formation of aggregates. See, e.g., Marrack, Annu. Rev. Microbiol. 9, 369-386 (1955). Furthermore, when the targets to be antagonized are on a cell-surface, binding of two cell surface targets by a single IgG may result in unwanted agonist activity via cross-linking or bringing together of the two molecules by the antibody. See, e.g., Prat et al., J. Cell. Sci. 111 9Pt2), 237-247 (1998). In addition, some full-size IgGs also exhibit poor penetration into tissues, especially solid tumors, and poor or absent binding to regions of some antigens that are occluded and can only be accessed by molecules of smaller size. See, Ying et al., J. Biol. Chem. (2012). Accordingly, in order to overcome the potential drawbacks associated with the bivalency of therapeutic antibodies and dimeric Fc fusion proteins, “one-armed” antibody, “one-armed” Fc fusion proteins, or a variety of antibody fragments of smaller size, have been recently explored for various therapeutic targets in order to improve a biological activity, bioavailability, and/or pharmacokinetics of therapeutic molecules. See, e.g., Demignot et al., Cancer Res. 50, 2936-2942 (1990), and Dumont et al., BioDrugs 20, 151-160 (2006). Thus, monomeric immunoglobulin Fc molecules, monovalent antibodies, and antibody Fc molecules have been described. See, e.g., US2006/0074225, WO2007/059782, WO2008/145139, WO2011/005621, and WO2011/063348. Despite the recognition that monomeric forms of antibodies and Fc molecules, and proteins comprising them, would provide certain advantages in development of therapeutic molecules, there remains a long-felt need for monomeric antibodies and fusion proteins which are stable but which do not exhibit increased immunogenicity or suffer from other drawbacks of the protein engineering required to achieve stable monomeric proteins.
N-glycosylation can have an impact on the protein stability, susceptibility to protease and immunogenicity as well as on the in vivo bioactivity of therapeutic proteins. See, e.g., Sola et al., J. Pharm. Sci. 98, 1223-1245 (2009), and Elliott et al., Nature Biotechnology 21, 414-421 (2003). Asparagine-linked glycosylation (Asn-linked or N-linked glycosylation) is one of the most common forms of post-translational modification of proteins in eukaryotic organisms. In general, the modification occurs at an asparagine residue in the first position of the consensus sequence of Asn-X-Ser/Thr, where the second position, “X”, is any amino acid except proline and wherein the third position is either serine or threonine such that Asn-X-Ser and Asn-X-Thr are considered canonical potential glycosylation sites in mammalian proteins. Shakin-Eshleman et al., J. Biol. Chem. 271, 6363-6366 (1996). Native human IgG antibodies have an N-glycan at Asn297 on the CH2 region of Fc domain. Crystal structures of the Fc domains have also revealed that the carbohydrates are packed within the internal space enclosed by the CH2 domain. While CH2 domains from two polypeptide chains make no direct interactions due to the carbohydrate moieties, the CH3 domains associate with each other through a large hydrophobic interface. Accordingly, it would be desirable to generate a stable monomeric form of a Fc domain with a prolonged in vivo half-life and other improved pharmacokinetics using the N-glycosylation engineering approach, in which the engineered glycan not only can separate the CH3-CH3 interface, but also can cover the exposed hydrophobic surface of CH3 domain to avoid aggregation and potential immunogenicity. The present invention fulfills this need.